The central hypothesis of this project is that tumor necrosis factor-alpha (TNF) is produced by the thick ascending limb of Henle's loop (TAL) and is part of an adaptive pathway that attenuates increases in blood pressure in response to high NaCl intake. The TAL-derived TNF inhibits Na+-K+-2Cl- cotransporter (NKCC2) expression and activity and is produced in response to high NaCl intake via a transcriptional mechanism involving NFAT5, also known as tonicity-responsive enchancer binding protein (TonEBP). Thus, TNF production by the TAL in response to high salt intake serves as a negative feedback mechanism to attenuate NKCC2 expression and activity, thereby contributing to the maintenance of blood pressure by limiting an increase in NaCl reabsorption. The maintenance of sodium homeostasis is essential for the regulation of blood pressure and insights regarding novel mechanisms that regulate renal sodium transporters could serve as a framework for development of new antihypertensive therapies. Three main objectives will pursued during this study: 1) Determine the effects of TAL-derived TNF on the blood pressure and renal functional responses to HS intake under normotensive/non-inflammatory conditions, 2) Define the mechanisms by which TNF inhibits NKCC2 expression and activity in the mTAL, and 3) Determine the role of NFAT5 as a regulator of the TAL- derived TNF production and responses to HS intake. TAL cell-specific silencing of TNF will be accomplished using purified lentivirus constructs under the control of a Tamm-Horsfall glycoprotein (THP)-specific promoter construct and by a transgenic mouse strategy to genetically delete TNF in the TAL using the Cre/loxP approach. These models will be used to evaluate the effects of TNF derived from the TAL on blood pressure, renal function and NKCC2 expression and activity under normal and high NaCl conditions. The approaches to study NKCC2 regulation by TNF include in vivo studies in parallel with freshly isolated mTAL tubules, which will be used for cellular determinations of NKCC2 phosphorylation, activity, and analysis of the signaling pathways that are inhibited by TNF. Primary cultures of polarized mouse mTAL cells also will be used to study the mechanisms by which TNF inhibits NKCC2 phosphorylation. TAL-specific lentivirus silencing of NFAT isoforms in vivo will be used to determine the mechanism by which TNF is produced in this segment of the nephron and the expression and translocation from NFAT isoforms from cytoplasm to nucleus will be determined in mTAL tubules and cells. This project will advance our understanding of a novel intrarenal regulatory pathway involving TNF, which contributes to blood pressure homeostasis in response to high salt intake.